Point-of-sampling detection of Lyme and other tick-borne diseases Foundation for Applied Molecular Evolution Steven A. Benner ABSTRACT Lyme disease is today the most prevalent vector-borne disease in the US, and ticks in general are the most significant vectors for human pathogens worldwide, including Borrelia, Babesia, and Rickettsia species. For Lyme disease, the CDC confirmed only 28,000 cases in a population that might actually hold 300,000. This severe underdiagnosis is due to: (a) expensive and spotty environmental surveillance, yielding insufficient patient and physician awareness, and (b) inadequate molecular tests; those exploiting antibodies can give 86% false negatives. Further, to order a molecular test (at $260), a physician must guess that a patient suffers from one of these diseases. As single pathogen-single tests are the norm in the FDA-regulated diagnostics space, these guesses must expand (each $200) to other candidate pathogens, one at a time. Not surprisingly, aggressive advocacy groups have arisen to counter (what they feel to be) inadequate CDC/NIH response to this disease threat. This R21 project will deliver a molecular test that, in one step in the hands of uncertified users, identifies a complete panel of all tick-borne pathogens, for less than $1.00, and within 30 minutes. The assay will apply to ticks directly, either trapped or pulled off a pet or a child. It will be useable by college nurses, doctors in their offices, public health service staff, and even laypeople, to examine ticks. After this R21 project is complete, the data will set the stage for a human diagnostic kit that can be FDA-approved to apply to patient samples. Although the Benner lab has never before worked with tick-borne diseases, this proposal has a high chance for success, as it has been de-risked by work, just published, where the lab delivered a kit that detects dengue, chikungunya, and/or Zika in a single assay, in 30 minutes, in a single mosquito carcass. The sample is rendered free of biohazard by treatment with ammonia (similar to Windex) and ethanol, and dropped into a pre- prepared tube containing dry reagents, shipped without refrigeration. Alternatively, urine or blood may be used. This kit is being used today to monitor the presence of pathogens in trapped mosquitoes, and shipped to China and India, where it detects dengue and chikungunya in human urine and blood. This illustrates our strategy of developing unregulated tests to support introduction of tests for patient use. The deliverable will be benchmarked on real ticks containing real pathogens, provided by the laboratory of Maria Diuk-Wasser at Columbia. This will include blind testing on ca. 50 samples. The premises are: (a) The NH3-EtOH disruption is sufficient to release detectable amounts of pathogen target; (b) multiplexing in a reverse-LAMP architecture can be increased from 3 to ~10 to cover all tick targets; and (c) AEGIS in displace- able probes will support a patterned readout for different pathogens, and a control. The authentication of the reagents will be assured by making the deliverable available to the public, as is being done for mosquito- borne pathogen detection.